JPS62244382A - Tryptophan operon, peptide and protein coded thereby, utilization of tryptophan operon gene expression and production of tryptophan - Google Patents

Abstract

PURPOSE:To efficiently produce the titled tryptophan in a large amount, by cultivating a microorganism transformed by a DNA sequence capable of coding an enzymic group of a tryptophan synthetic system, etc., in a culture medium. CONSTITUTION:A chromosomic gene obtained by extracting microbial cells of Brevibacterium lactofermentum, etc., of the genus Brevibacterium is cleaved with a restriction enzyme to give a DNA, consisting of DNA expressed by the formula containing an operator region controlling the synthesis of m-RNA, promoter region controlling the synthesis of m-RAN, etc. The resultant DNA is then subjected to ligation reaction with a plasmid vector to afford a recombinant DNA, which is further introduced into a variant strain requiring tryptophan to carry out transformation and provide coryneform bacteria having the ability to produce tryptophan. The resultant bacteria are then aerobically cultivated in a culture medium containing a carbon source, nitrogen source, etc., to produce and accumulate the aimed tryptophan in the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of coryneform bacteria, more specifically Brevibacterium, which can be applied to the molecular breeding of L-1-lyptophan-producing bacteria and the production of physiologically active peptides by recombinant DNN methods. Regarding the tryptophan operon sequence of U.

Coryneform bacteria (C'oryneform) according to the present invention
bacteria) is described in Percy's Manual of Determinative Bacteriology (Barge
ysManual of Determinative
Bacteriology) 8th edition 599 pages (19
It is a group of microorganisms defined in 7 4), and is an aerobic, Durham-positive, non-acid fasting bacillus that does not have spore-forming ability. Among these coryneform bacteria, the following coryneform glutamate-producing bacteria are particularly preferred in the present invention.

Examples of wild strains of coryneform glutamate-producing bacteria include the following.

To Brevibacterium divaricatum TCC 14020 Brevibacterium nasocalolyticum ATCC14
066 TCC14 to Prebihacterium inmariophyllum
068 Flevipacterium lactofermentum ATCC1
3869 Verbihacterium roserum ATCC13825
Frechybacterium flavum ATCC13826
To Brevihacterium thiogenitalis TCC19240 Corynebacterium acetoacidophilum 8TCC1
3870 Corynebacterium acetoglutamine ATCC15
806 Corynebacterium carnae 8TCC15991 Corynebacterium glutamic acid ATCC13032,13060 Corynebacterium Lilium ATCC15990 Corynebacterium melasecola ATCC17965 Microbacterium ammoniaphyllum to TCC15
354 The coryneform glutamate-producing bacteria of the present invention include not only wild strains having glutamate productivity as described above, but also mutant strains having glutamate productivity or having lost glutamate productivity.

The tryptophan operon here includes a promoter, an attenuator, a leader region (trpL) encoding a leader peptide, anthranilate synthase genes (trpE, trpG), and a phosphoribosylanthranilate transferase gene (trpD).
), N-(5'-phosphoribosyl) anthranilate inmerase indole-3-glycerol phosphate synthase gene (trpC), and) liptophan synthase genes (trpB, trpA) are arranged adjacently, A transcription unit that functions as one transcription unit.

The method for isolating each structural gene is to first extract a chromosomal gene from a tryptophan operon of a coryneform bacterium or a strain having each structural gene, for example, 11.5a.
ito and, Miura Biochem, Bi
ophys.

The method of Uta 72, 619 (1963) can be used. ), which is then cut with an appropriate restriction enzyme. Next, it is connected to a vector that can replicate in microbial cells and has promoter activity, and the resulting recombinant DNA is used to mutate the structural gene of the tryptophan biosynthesis system in coryneform bacteria or other microorganisms. , by transforming a mutant strain in which the enzyme does not lose its activity and therefore exhibits tryptophan auxotrophy, the enzyme activity is restored or increased,
A strain in which the tryptophan requirement disappears is collected, and a complex plasmid containing the structural gene can be isolated from this strain.

Even with this method, if you are lucky enough to cover the entire operon with 'i'
However, if the entire operon cannot be isolated (cloned), label part or all of each structural gene isolated using the above method with an isotope, etc., and use it as a probe. It can be isolated by colony hybridization from a chromosomal gene scene bank of coryneform bacteria created using a plasmid or a phage/lid.

To cleave chromosomal genes, a wide variety of restriction enzymes can be used by adjusting the degree of cleavage by adjusting the cleavage reaction time, etc.

In the present invention, the vector used when the tryptophan operon or a part thereof is used for the production of tryptophan can be used in coryneform bacterial cells or in E, Goli, B
, 5ubLilis may be used as long as it can proliferate. Specific examples include the following.

(11pAM 330 JP-A-58-67699 reference (21pAM 1519 JP-A-58-77895 reference (3) pAJ 655 JP-A-58-192900
Reference (4) pAJ 611 Same as above (511) AJ 1844 Same as above (6) pCG 1 Reference to JP-A-57-134500 (7j pCG 2 Reference to JP-A-58-35197 (81pCG4 Reference to JP-A-57-183799 (91pcc 11 Same as above) 10) pCC1 Unpublished (Mriutin/A
jico) (11) pBL 100 Unpublished
(~) (12) pBR322 (13) pC194 Hector DNA can be cleaved by using a restriction enzyme that cuts the DNA at one site, or by cutting at multiple sites.
Partial cleavage is carried out using restriction enzymes.

The hector DNA is cut by the restriction enzyme used to cut the chromosomal gene, or oligonucleotides having complementary base sequences are connected to both ends of the cut chromosomal DNA fragment and the cut hector DNA, and Next, 1 hectare of gelatin and chromosomal DNA fragments are subjected to a lysis reaction.

The thus obtained recombinant chromosomal DNA and Hector DNA are transferred to recipient bacteria that succumb to coryneform bacteria'), 7
f-person, as reported by Escherichia coli for -12 (Mandel, M. and I.
liga.

8,,J,Mo1. , Biol,, 53.159 (
(1970) treated recipient cells with calcium chloride to produce DN.
A method to increase the permeability of Bacillus subtilis, or as reported for Bacillus subtilis (Duncan, C.H.,
Wilson, G., and Young, F.E.
,, Gene, Eng., 153 (1977)) t(Il
This is possible by 1) bringing the cells to a proliferation stage (so-called Combiten I cells) in which they become capable of taking up NA;

Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Chan [X, S, a
ndChoen, S.N., Molec, Gen.
, Genet, 368.111 (1979); Bi
bb, M, J, Jard, J, M, and ll
opwood, O, 8, 1 Nature, γ74.
398<1978); Hinnen, A., l1ick
s, J, B.

and Fr1nk, G, R,, Proc, NaLl
, Δcad, sci, UsA, Etou 1929 (1978
)) It is also possible to introduce the recombinant DNA into the recipient bacteria in the form of protoplasts or spheroplasts, which readily incorporate into the plasmid F'DN.

In the protoplast method, it is possible to obtain a sufficiently high frequency using the method used for Bacillus subtilis mentioned above, and polyethylene glycol or polyvinyl Of course, a method of incorporating DNA in the presence of alcohol and divalent metal ions can also be used. Equivalent results can also be obtained by adding carboxymethyl cellulose, dextran, Ficoll, Pluronic F68 (Selva), etc. in place of polyethylene glycol or polyvinyl alcohol to promote uptake into ON.

To carry out molecular breeding of tryptophan-producing bacteria using the cloned tryptophan operon or each structural gene, a strain transformed with the tryptophan auxotrophic mutant strain used for gene cloning can be used as a host. If the host shown below is used, a strain with higher tryptophan productivity may be obtained.

Mutant strains of Brevibacterium that require phenylalanine and tyrosine and are resistant to 5-methyltryptophan (1,5hiio, 11.5ato, M, Nakag
Wow.

Agric, Biol, Chem, 36.2315
(1972)), a mutant strain of Brevibacterium flexi that requires phenylalanine and is resistant to m-fluorophenylalanine and 5-fluorotryptophan (r, 5h
iio, S.

Sugimoto, M., Nakagawa, , Agri.
c, Biol, Chemi,, 39+627 (19
75) ), a mutant strain of Brevibacterium that requires tyrosine and is resistant to 5-fluorotryptophan and aza'serine, a mutant strain of Corynebacterium that requires phenylalanine and tyrosine, and is resistant to 5-methyltryptophan, 4
- Mutant strain resistant to methyltryptophan, 6-fluorotryptophan, tributophane hydroxamate 1-1p-fluorophenylalanine, tyrosine hydroxamate, and phenylalanine hydroxamate (Il
, Hagino.

, Nakagawa, 5ric to +, Bio1. Ch
Em., 39.345 (1975)).

The method of culturing the thus obtained coryneform bacteria capable of producing tryptophan to produce and accumulate tryptophan is not particularly different from the method conventionally used for the production of tryptophan using coryneform bacteria. . That is, the culture medium contains carbon sources, nitrogen sources, inorganic ions, and organic Eaf such as amino acids and vitamins as necessary.
It is the usual one containing fl nutrients. As the carbon source, glucose, sucrose, lactose, etc., starch hydrolyzate containing these, whey, θg honey, etc. are used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, etc. can be used.

When culturing, adjust the ρ11 and temperature of the medium to 3 J under aerobic conditions.
! As described in Section II, the process is continued until the production and accumulation of tryptopide-77 is substantially stopped.

In addition to the molecular breeding of tryptophan-producing bacteria, another great advantage obtained by the present invention is that the promoter of the tryptophan operon of Brevibacterium has the promoter of the tryptophan operon of E. coli and its trailing It has a strong terminator, as expected from its structure, and also contains an operator whose expression is regulated by tryptophan in coli form bacteria. E. coli
Expression of a heterologous gene, such as ink-ferrin, growth hormone, interleukin, nerve growth factor, or other bioactive polypeptides or enzymes, or overproduction of a heterologous protein in E. The E. coli l-ributophane promoter, operator, and terminator is frequently used. That is, the tryptophan operon obtained by the present invention naturally promotes the molecular breeding of tryptophan-producing bacteria in coryneform type bacteria, but it also promotes the molecular breeding of tryptophan-producing bacteria in coryneform bacteria. It has promoters, operators, humans, and terminators that can strongly express and regulate genes in E. coli or other coryneform bacteria, and this DNA sequence can be used to strongly express the above-mentioned heterologous genes. It is possible to express and overproduce.

Furthermore, among the DNA sequences of the present invention, promoter regions, operator regions, which are parts involved in gene expression,
When the base sequences of the attenuator region, liposome binding region, and terminator region are used alone or in combination (taken out), or when the base sequences of the structural genes of each enzyme are used. Furthermore, DNA sequences obtained by substituting sequences so that the encoded amino acid sequences do not differ are also included in the D of the present invention.
Substituting bases in any part of the NA sequence with others,
When a new base is inserted or deleted, or
It is assumed that both the derivative obtained by transposing a part of the base sequence and the protein of the amino acid sequence encoded by it provide gene expression and L-tryptophan, and the main part depends on the present invention. This technology falls within the scope of the present invention.

Hereinafter, specific examples will be given of a method for obtaining the top 1-fan operon of Prehybacterium lactofermenta J containing the DNA sequence of the present invention, determination of the base sequence and amino acid sequence of the DNA of the present invention, and the present invention. L-1 produced by Coryneform bacteria obtained by transformation using ON8 of
- Example 1 describing production of liptophan, promoter and operator.

Cloning of anthranilate synthase gene, phosphoribosyl anthranilate transferase gene, and tryptophan synthase β subunit gene 1-1 Preparation of chromosomal DNA containing the tryptophan operon of Brevibacterium lactofermentum J112
25 (FIERM-P4370) in 1β CI'lG medium (peptone 1 g/d1, yeast extract Ig/d1, glucose 0, 5 g/d1, and NaC6 0.5 g
/di and adjusted to pH 7.2), cultured with shaking at 30°C for about 3 hours, and the cells in the logarithmic growth phase were collected.

After lysing this bacterial cell with lysozyme/SOS, the chromosomal DNA is extracted and purified using the usual phenol treatment method, and finally 3. 5 mg of DNA was obtained.

1-2 Preparation of Vector ON Using ρ^J184/l (molecule: 15.4 megadaltons) as a vector, its DNA was prepared as follows.

First, Lactonorcimentum August 2037, which holds face seal 844 as plasmid 1'
was inoculated into 100ml of CMG medium and grown logarithmically at 30℃! After culturing to IJ1 to L1, the cells were lysed by lysozyme SDS treatment, and a supernatant was obtained by ultracentrifugation at 30,000×g for 30 minutes. After the phenol treatment, 2 volumes of ethanol were added to precipitate and collect the DNA. This was mixed with a small amount of TEN buffer (20mM Tris-HCl, 20mM
hl! , 1mM [EDTA (pH 8,0)
), the plasmid fraction was separated by cesium chloride-edidium bromide density gradient equilibrium centrifugation, and finally p
Approximately 200 μg of AJ 1844 plasmid DNA was obtained.

Insertion of La S envelope DNA fragment into vector 10 μg of chromosomal DNA obtained in 1-1 and 10 μg of chromosomal DNA obtained in 1-2
5 μg of NA and restriction endonuclease Pst I
Each was held at 37°C for 1 hour and cut. 65°
After heating at C for 10 minutes, both reaction solutions were mixed and the T4 phage-derived DNA
The DNA strands were ligated by holding at 10°C for 24 hours using ligase. The reaction solution was then heated at 65°C for 5 minutes, and 2 volumes of ethanol was added to the reaction solution to collect the ligated DNA precipitate.

1-4 Cloning of anthranilate syncose gene, phosphoribosylanthranilate transferase gene, and tryptophan synthase β subunit gene Brevibacterium lactofermentum anthranilate synthase-deficient strain AS60, phosphoribosylanthranilate transferase-deficient strain Nc38, tryptophan synthase β subunit deletion strain 11kL30
(Both use J12125 as the parent strain, and N-methyl-N
-nitro-N-21-isoguanidine) was used as the DNA recipient.

The protoplast transformation method was used as the transformation method. First, transfer the bacterial strain to 5 ml of CMG.
Cultured in liquid medium until early logarithmic growth phase, then treated with penicillin G.
After adding 7 ml of 0°6 UniSoto, culture with shaking for another 1.5 hours, collect the bacterial cells by centrifugation, and reduce the bacterial cells to 0.5 M.
Sucrose, 20-maleic acid, 20mM magnesium chloride, 3.5% Bena-7 Sabros (Dirco)
SMMP medium (pl+ 6.5) consisting of 0.5mf
Washed with f. Next, the cells were suspended in an SMMP medium containing Lmg/mA norizozyme and allowed to undergo pro1-plast formation at 30°C for 20 hours. Centrifuge at 6000xg for 10 minutes xtt
1&, protoplasts were washed with SMMP and 0.5 m/
, S) resuspended in IMP. The protoplasts thus obtained were mixed in the presence of 5mM EDTA, polyethylene glycol was added to a final concentration of 30%, and then the DNA
The cells were left at room temperature for 2 minutes to allow them to be incorporated into protoplasts. The protoplasts were placed in 1 m of SMMf' medium.
After washing with e.g., the cells were resuspended in 1 ml of SMMP medium and cultured at 30''C for 2 hours for expression. This culture solution was spread on protoplast regeneration medium at pl+7.0.

The protoplast regeneration medium is 1-Lis(
1 2 g of hydroxymethyl)aminomethane, X(1
0.5 g, glucose 10g1MgCAz・6tl
zO 8.1 g, CaCjl! z・2Hz0 2
．． 2 g, peptone 4 g, powdered yeast extract 4 g, casamino acid (Difco) 1 g, K2HP
O4.0. 2 g, sodium succinate 135 g, agar 8 g and chloramphenicol 3 μg 7 m I! including.

After two weeks of incubation at 30°C, each recipient bacterium was incubated with &']2
5,000 chloramphenicol-resistant colonies appeared, which were then transferred to minimal medium (2% glucose, 1%
Ammonium sulfate, 0.3% urea, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 2
ppm iron ion, 2 ppm manganese ion, 200μ
g/l thiamine hydrochloride, 50°C g/l biotin, casamino acids (Dirco) 3 g/7! , chloramphenicol 10 μg/ln (1, pH 7, O
, agar 1.8%), and the strain that is chloramphenicol resistant and loses tryptophan auxotrophy was transformed into AS.
Two strains were obtained from the classification using 60, one strain from the classification using llh3'8, and one strain from the classification using Kan30.

When plasmids were extracted from the five strains mentioned above, all of the plasmids were clearly larger than the Hector plasmid pAJ1844.
Me breath 1' ptrplE36, pLrp[i
4. The recombinant plasmids obtained from the partitioning using Inhibi 38 were
3 obtained from classification using trp03851°No.30
(2) The replacement plasmid was named pLrpB301.

1-57f￥ Recombinant plasmid ptrp[i36, pmurpE4, pt, rpD3851, ptrpB301 obtained in Transformation 14
To confirm the presence of the anthranilate synthase gene, phosphoribosyl anthranilate transferase gene, and tryptophan synthase β subunit gene, respectively, ptrpE36 and ptrpE4 were placed in AS60, ptrpl) 3851 was placed in floor 38, and ptr
pB301 was retransferred to NO. 30.

Ten chloramphenylcol-resistant colonies were picked up and their tryptophan requirement was examined. As a result, all of them lost their requirement, and pLr
pH36, ptrpE4 contains the anthranilate synthase gene, ptrpD3851 contains the phosphoribosyl anthranilate transferase gene, ptr
It was revealed that pB301 contains the tryptophan synthase β nabuunisoto gene. However, pt.
In the rpE4 transformed strain, the degree of loss of auxotrophy and the accumulation of anthranilic acid on the minimal medium were determined by p trpE4.
It was suggested that ptrpE4 may lack part of the anthranilate synthase gene compared to the 36 transformed strains.

1-6 Preparation of restriction enzyme map of inserted DNA fragment of Ml recombinant plasmid Recombinant plasmid pL was created by the method used in Example 1-2.
rpE36, ptrp[E4, ptrpD3851
, ptrpB301 was prepared three times and cut with various restriction enzymes according to conventional methods to prepare a restriction enzyme map of the inserted DNA fragment (Fig. 1).

Example 2 Tryptophan operon of Brevibacterium lactofermentum Brevibacterium lactofermentum AJ1122
Chromosomal D
Prepare NA, use restriction enzyme Bamll or Sail,
or complete cleavage with Xhol and E. E.coli vector pLJ (18) (Messing, J., et al.
, Gene.

■, 103-]. L9 (1985)) into each restriction enzyme cleavage site, [i. coli J old 09 (Messi
ng, J. , et al. +Gene+33+103-
119 (1985)) and transformed X-Gal (5
-bromo-4chloro-3- indoly+
- β-galactoside), IPTG (i
Sopropy '-β-D-thio-galac
topyranoside) and ampicillin and plated on agar medium. After culturing at 37°C for 24 hours, a total of about 1,500 white colonies appeared in 2 liters.
fished onto a cellulose filter. 1.2 kb of ptrpE36 containing the anthranilate synthase gene (trp[i) obtained in Example 1. Colony hybridization (Grunstein.

M. , Walls, J. :Methods in Ii
nzymoloBy, starvation. 379, Δacademic
Press Inc. +New York (1979)
), and one positive clone was obtained from the section using the control K11 enzyme BamH I and one positive clone was obtained from the section using the restriction enzyme Sall. [The recombinant plasmid obtained from the lamH 1 section was named ptrpE97, and the plasmid obtained from the Sa11 section was named ptrpE42, Example 1
It was revealed that the inserted Pstl fragment of ptrpB301 had the same restriction enzyme map as the inserted Pstl fragment using the method shown in .

In addition, p trpE97 and ptrpE42 have a common Bam
) II-Sall fragment.

Example 3 Subcloning of the N-(5-phospholiposyl)anthranilate inmerase indole-3-glycerol phosphate synthase gene (trpc) and subcloning of the tryptophan synthase subunit gene (trpA) The recombinant plasmid shown in FIG. insert DNA
The trpD gene and trp were identified by comparing the restriction enzyme maps of the fragments.
It was thought that the trpC gene exists between the B genes, and the trpA gene exists downstream of the trpB gene. Therefore, the following experiment was conducted to confirm the existence of each gene.

3-1 Subcloning of trpcl gene Approximately 2 kb shown in Figure 1 from recombinant plasmid p trpE97
The Sst[-1EcoRI fragment of , 5stl,
pLIC19 (Messing,
J,,et al,,Gene, 33°103-1!
9.1985) and arranged to enable transcription from the Kenji promoter. Or about 2.6 in Figure 1
The 5stl-11ind m fragment of kb was fractionated into Ss.

pUclB (Messin) cut with tlindlII
B, J,, at al, +Gene.

η, 103-119.1985)), positioned to allow transcription from the h rainbow promoter, E,
coli results, 5stl-EcoRI fragment, or 5s
The recombinant plasmid containing the jl heat find Ui fragment abolished the auxotrophy of IE and coli.

3-2 Confirmation of existence of trpA gene The approximately 2.4 kb Nrur-namllT fragment shown in FIG. 1 was fractionated from the recombinant plasmid ptrpE97, and was separated into Smal.

[pU cut with 1amll+ (ligated to , 18, Ia
arranged so that transcription from the c promoter is possible,
ε, coli CGSCNo., 5644 (t
+ rha-L λ −) was transformed into rp83. As a result, in the transformed strain carrying the recombinant plasmid containing the Nrul-tlamllll fragment,
Disappearance of Toriji 1-Fan requirement was observed.

Example 4 Determination of base sequence of tryptophan operon ptrpE36, ptrpD3851, ptr obtained in Example 1
Plasmids were prepared from each of the transformants having pB301 and ptrpE97 obtained in Example 2.

For each plasmid insert DNA fragment, UCl3
or pU(, 19 or old 3mplO(Messing
, J., and Vicira.

J. Gen. 9.269 (1982)) using the dideoxy chain termination method (Sanger, F., eL al., Proc., Nat.
l.

Acad, Sci, USA74.5463 (1977)
), the entire nucleotide sequence of the 1-ributophane operon was determined according to the strategy diagram for nucleotide sequence determination shown in FIG. As a result, the following DNA base sequence was obtained, and this base sequence contains the RNA polymerase binding site (trp promoter) necessary for the expression of the tryptophan operon of Brevibacterium lactofermentum, the ribosome binding site, and the anthranilate syncusase gene. (jrpl
E, trpG), phosphoribosyl anthranilate transferase gene (trpD), N-(5-phosphoribosyl) anthranilate isomerase indole-3-glycerol phosphate synthase gene (trpC
), tryptophan synthase gene (trpH, tr
pA) and a termination sequence (terminator).

Furthermore, between the promoter and the trpE structural gene, there is an operator region involved in the expression regulation mechanism at the transcriptional level, reprej.
pL) and a region in which an attenuator-like structure exists (Fig. 3). The structure of the terminator is shown in FIG.

Example 5 Promoter Isolation and Activity Confirmation As a result of the nucleotide sequence determination, the putative tryptophan operon promoter was isolated, and in order to confirm its function, ptrpE97 or ptrpE97 or pt
EcoRI-Hind III fragment of rpE36 (approximately 5
50 bp, ) to E. coli promoter probe vector pkk+'75-6 (7 nubicillin resistant (Ap
), tetracycline (Tc) sensitive) (Brosiu
Gene 27, 15H1984)). The obtained recombinant plasmid ptrpPO
1 expressed Tc resistance in E. coli.

Furthermore, we added Pst to the PstT cleavage site of pAJ226, a 94M330-derived trif1-prim resistant vector.
When Brevibacterium AJ11225 was transformed with the ligated recombinant plasmid ptrpPO1 cut with l, expression of Tc resistance was observed. As shown in Table 1, the Tc resistance of the transformant having this recombinant plasmid ptrρPO2 increased in the presence of Trp. Therefore, it is thought that a promoter and an operator exist in this region.

Next, to further restrict the promoter region, the resulting Alul-11ind'M fragment (51bP,) and 1
It was revealed that a promoter exists on lael-11ind III (135 bp). Similar results were obtained with E. coli promoter probe Hector pK.
K232-8 (Ap resistant, chloramphenicol sensitive) was used, and it was confirmed that the promoter was present on the Alul-11indlII fragment (51 bp).

Example 6 Phosphoribosyl anthranilate 1-transferase gene (trpD), N-(5-phosphoribosyl) anthranilate isomerase indole-3-glycerol phosphate synthase gene (LrpC), l-liptophan synthase gene (trpH Breeding of tryptophan-producing bacteria by amplification of , trpA).

trpD of the cloned Previbacterium lactofermentum tryptophan oberon.

L-tryptophan production was investigated using plasmid I pAJ234, which has 4 genes: trpC, trpB, and trpA.

Transformation was performed using pAJ234 using the m-fluorophenylalanine method, and transformed strains were selected using chloramphenicol resistance as an indicator. The thus obtained 8J12195 (FERM-P4O10) was cultured and its tryptophan production ability was examined, and the results shown in Table 2 were obtained.

Culture was performed using tryptophan production medium (glucose 130 g
, (Nl14.)zsOa 25 g, fumaric acid 12 g.

3 ml acetic acid, KHzP041 g, Mn5Oa'
711zO10mg, MgSO4・711□O1g,
d-Biotin 50μg 1 Cyane hydrochloride 2000μg
, methionine 400■, thiillin 650mg, soybean protein acid hydrolyzate "Ajiri" 50ml, CaC0:+
50 g in 1β water, pl + 6.5°) 20mf
The test bacterial strain was inoculated into a 500 mj+ Sakalo flask and incubated at 30°C for 72 hours with shaking. After culturing, L-1 lymphophan in the centrifuged supernatant was extracted from Leuconostoc mesenteroides (Leuconosto mesenteroides).
c mesenteroides) 8 TCC8042 was determined by a bioassay method using as a quantitative strain.

Table 2 Accumulation amount of L-toIJ butophane in transformed strain In order to obtain M247, deposited AJ 12195
It is possible to remove complex plasmids in host cells without damaging the host cells. That is, the plasmid can be naturally lost from the host, or it can be removed by a "removal" operation (Bact, Rev., 36).
．． p3 (i!-405 (1972)). Examples of other removal operations are as follows. AJ 12195 was inoculated in a CMG liquid 1 field, cultured overnight at 37°C (high temperature treatment), the culture solution was diluted appropriately, and applied to a CMG agar medium containing or not containing chloramphenicol. One strain isolated as being susceptible to call is M247.

[Brief explanation of drawings]

Figure 1: Restriction enzyme map of the DNA fragment inserted into the AM +A plasmid Figure 2: Strategy diagram of the tryptophan operon of Brevibacterium lactofermentum
. Figure 3: Tryptophan operon of Brevibacterium lactofermentum - LrpE of Brevibacterium lactofermentum, cloned into pLIcl 9 or old 3mplO, and the base sequence determined by the dideoxy method in the direction indicated by the arrow.
Base sequence of the 5' upstream region of the structural gene, deduced amino acid sequence, and predicted secondary structure to RN - Figure 4 Structure, promoter, and operator region of the tryptophan operon of Brevibacterium lactofermenta l. Figure 5. Strategy for isolation and identification of Brevibacterium lactofermentum tryptophan operon structure and strategy for defining promoter and operator regions -35 and -10 are [E. Figure 6 shows the regions corresponding to the -35 and -10Tf4 regions of the coli promoter consensus sequence. Structure of the terminator of the tryptophan operon of Brevibacterium lactofermentum.

Claims (1)

[Claims] 1) An operator region that controls m-RNA synthesis, a promoter region that controls m-RNA synthesis, an attenuator region that controls m-RNA synthesis, and ribosomes necessary for protein synthesis. m-RN
A binding region, a region encoding a leader peptide,
DNA formula 1 consists of the sequence shown in Formula 1 below, which includes a region encoding the enzyme group of the tryptophan synthesis system and a terminator region that forms a signal to stop synthesis of m-RNA at the end [There is a gene sequence] 2) DNA consisting of one or more selected from the group consisting of various regions described in claim 1. 3) The DNA according to claim 1 or 2, wherein the DNA is artificially synthesized DNA or DNA derived from a microorganism. 4) The DNA according to claim 3, wherein the microorganism is a coryneform bacterium. 5) The DNA according to claim 3, wherein the microorganism belongs to the genus Brevibacterium. 6) D according to claim 3, wherein the microorganism is a microorganism belonging to Brevibacterium lactofermentum.
N.A. 7) The DNA according to claims 1 to 6, wherein the DNA is partially substituted, mutated, or deleted. 8) The DNA according to claims 1 to 7, wherein the DNA is DNA integrated into a plasmid or a phage-derived vector. 9) DNA according to claim 2 and or 8
A method for producing L-tryptophan using 10) Peptides or proteins having the amino acid sequences of formulas 2 to 7 below (A alanine, C
Cysteine, D-aspartic acid, E-glutamic acid, F-phenylalanine, G-glycine, H-histidine, I-isoleucine, K-lysine, L-leucine, M-methionine, N-asparagine, P-proline, Q-glutamine, R-arginine,
Indicates S serine, T threonine, V valine, W tryptophan, and Y tyrosine. In addition, the second formula is tryp-E enzyme, the third formula is tryp-G enzyme,
The fourth formula shows the amino acid sequence of tryp D enzyme, the fifth formula shows the tryp C enzyme, the sixth formula shows the tryp B enzyme, and the seventh formula shows the tryp A enzyme. ). Formula 2 [There is an amino acid sequence] Formula 3 [There is an amino acid sequence] Formula 4 [There is an amino acid sequence] Formula 5 [There is an amino acid sequence] Formula 6 [There is an amino acid sequence] Formula 7 [There is an amino acid sequence] 11) The peptide or protein according to claim 10, wherein the amino acid sequence is partially substituted, mutated, or deleted. 12) DNA encoding the peptide or protein according to claims 10 to 11. 13) A gene expression method using DNA having the promoter region sequence according to claim 1 and/or claim 7. 14) A gene expression method using DNA having the operator region sequence according to claim 1 and/or claim 7. 15) A gene expression method using DNA having the sequence of the attenuator and/or leader peptide region according to claim 1 and/or claim 7. 16) A gene expression method using DNA having the terminator region sequence according to claim 1 and/or claim 7.